Our major goal in this proposal is to use novel culture conditions to achieve high-frequency selection and proliferation of transformed cells from (a) clinical specimens of breast carcinoma, and (b) cultures of human mammary epithelial cells (HMEC) after exposure to chemical carcinogens. Limitations in presently used protocols have significantly hampered progress in this area. Our novel approach will be to expose the mixture of normal cells and putative transformants (which is inevitable in both (a) and (b)above) to conditions which closely simulate the microenvironment of solid tumors. In this regard, the literature provides strong suggestive evidence that, due to an imbalance of tumor growth and vascularization, gradients of O2 and nutrients are formed even during very early stages of neoplasia. We hypothesize that neoplastic or transformed cells may harbor genetic and/or epigenetic changes which enable them to survive under these conditions and to acquire progressive alterations essential for subsequent stages of tumor progression. In culture, this altered responsiveness to the environment could provide the putative transformants selective expansion advantage over the surrounding normal cells, much the same way as it is thought to occur in vivo. We will test our hypothesis in a culture system in which anchorage-dependent cells can be subjected to self-created gradients of O2 and glucose. Moreover, we will determine relative transformation frequency in immunomagnetically separated populations of the luminal and basal subsets of the human mammary gland as well as in HMEC from ductal versus lobular areas of the mammary tree. We will use this novel culture system to ask whether the yield of experimentally-induced transformation is higher in HMEC derived from non-malignant mastectomy tissue including specimens from patients at a high risk of familial breast cancer. The innovative approach proposed in this application can provide an invaluable framework for the selective isolation of tumor cells from primary breast carcinoma tissue in order to facilitate studies on the early stages of malignant progression. Additionally, it will provide the opportunity to delineate markers that represent specific stages of in vitro and in vivo mammary epithelial transformation and to identify the most susceptible target cells. Most importantly, this study may provide a rapid means for investigating which chemical or physical agents alone or in combination are able to enhance or reduce transformation frequency, thereby facilitating studies on the etiology and mechanisms of carcinogenesis in epithelial cells of other tissues as well.